There are two major goals of this study. The first goal is to characterize key components of the basic myosin motor mechanism. Our second goal to begin the kinetic assessment of other members of the myosin family. We will extend this to include the roles of various trompomyosin isoforms in altering the kinetics of various members of the myosin family. We will begin a detailed kinetic assessment of other members of the myosin family with myosin V. Based on our preliminary data we hypothesize that myosin V, and likely other myosin family members have kinetics that are fundamentally different from myosin II. These kinetic differences may allow these motors to work either alone or in small numbers. This project will utilize in vitro expression and functional assays that will enable structure/function studies of recombinant Dictyostelium myosin II and chicken myosin V. Expression of enzymatically active fragments (S1-like and heavy meromyosin-like fragments) of myosin will be accomplished with the baculovirus/SF9 cell system. Functional evaluation of the expressed myosin will include ATPase measurements, determination of enzyme kinetic parameters and in vitro motility (translocation of actin filaments by myosin). Low resolution structures of the recombinant myosins will be examined using three-dimensional reconstructions of cryo-electron micrographs derived from S1-decorated actin filaments. In this manner we will begin to dissect the functional and structural domains of the myosin motor, within the framework of delineating the principles of myosin design and isoform diversity.